In one aspect the present invention relates to methods for performing chemical assays using photon counting. More particularly, the invention relates to the quantitative clinical chemistry determinations of very low concentrations of substances in biological media by measurement of fluorescence. In addition to fluorescence, other light emitting processes may be used, such as phosphorescence, luminescence induced by x-rays or radionuclide decay, or chemical luminescence. In another apsect this invention relates to fluorescently labeled compounds and their use in competitive binding techniques.
Fluorescence is the physical process whereby many substances absorb light of a given wave length which excites one or more atomic electrons in the material to a higher energy level. This excitation is followed after several nanoseconds by the return of the electrons to their normal energy level, accompanied by the emission of light of a higher wave length. The wave lengths of exciting light and of emitted light in the process of fluorescence are characteristic of each substance under consideration. Fluorescence can be produced by other kinds of electromagnetic radiation such as X-rays and radionuclide decay. Phosphorescence is a related process in which the excited electron reverses spin. The process of electron energy decay in phosphorescence is slow, and light is emitted over a period as long as seconds or minutes after excitation. Luminescence is another related process in which a chemical reaction causes excitation of an atomic electron followed by light emission.
Heretofore, fluorimeters used in quantifying all of these processes have measured the flux or rate of emitted light; that is, the continuous intensity of light emitted by the substance to be analyzed. The fluorimeters heretofore used in fluorescent analyses frequently made use of filters or monochromators to limit the bands of exciting and/or emitted light to those absorbed or emitted by the substance. The methods of measuring fluorescence by these flux-type fluorimeters are well known to the art and are described in many publications, for example, F. R. Elevitch, Fluorometric Techniques In Clinical Chemistry, Boston: Little, Brown & Co. (1973).
A second type of instrumentation which can be employed to quantify the light emitted by fluorescent compounds is a photon counter. Until recently photon counters were not employed in clinical assay techniques both because of their expense and because simple photon counting, without modification for use in connection with clinical sample materials, will not provide significantly improved results over those obtainable with a fluorimeter. Thus, for clinical determinations of the amount of a substance present in low concentrations in samples of biological fluid, fluorimeters taking the above described flux-type measurements have been employed exclusively.
A type of assay which is very extensively used in clinical chemistry determinations is the radio-immunoassay. This assay is used primarily for the quantification of hormones, drugs, metabolites, organic pollutants, and other biological or biochemical substances which are present in samples in very low concentrations. The radio-immunoassay is particularly suited to chemical determinations where great sensitivity and specificity are required.
The radio-immunoassay is a member of the class of chemical determinations referred to as competitive binding assays. Competitive binding assays are typified by the immune reactions between antigens and antibodies, and may be represented for example by the following formulas: ##EQU1## wherein A is the antigen in the unknown serum to be measured, A' is the same antigen labeled with a radioactive entity, B is a known antibody which is specific to the antigen (both to A and A'), and each k represents a reaction rate, and the ratio between k.sub.1 and k.sub.1.sup.' and between k.sub.2 and k.sub.2.sup.' can be determined.
Hence, when A, A' and B are placed together in a reaction medium, the antigens A and A' will compete for the binding sites on the antibody B so that some of the antigen-antibody complex will be unlabeled (AB) and some of the antigen antibody complex will be labeled (A'B). If the concentrations of A' and B are known, it then becomes a simple matter to determine the concentration of A, once the antigen-antibody complexes are separated from the reaction mixture. Thus, the radioactivity, and therefore the concentration of A', can be measured in either the fraction containing the antigen-antibody complexes or the fraction containing the remaining unreacted reaction mixture. The concentration of A is then inversely related to the radioactivity in the complexes and directly related to the radioactivity remaining in the unreacted reaction mixture. Any combination of ligand and specific binding protein can substitute for the antigen and its specific antibody.
Essentially, a simple radio-immunoassay is performed in the following manner. First, an aliquot of solution containing an unknown quantity of antigen or ligand is incubated with a known quantity of specific antibody or specific binding protein plus a known quantity of antigen or ligand labeled with a radioactive atom. The unlabeled, unknown antigen or ligand and the radioactivity labeled antigen or ligand compete with each other for binding sites on the antibody. After incubation, the antigen-antibody complexes (partly unlabeled and partly labeled) are separated from unbound antigen by any of a number of well known techniques, such as covalently bonding the antibody to or adsorbing the antibody on any of various substrates, such as activated charcoal, glass, plastic beads, test tube walls, etc. The radioactivity of either the bound or unbound fraction is determined, and the amount of unlabeled antigen may then be calculated or determined from a standard curve made up by doing sample runs using known amounts of unlabeled antigen. The performance of simple radio-immunoassays is described by J. Murphy, J. Clin. Endocrinol, 27, 973 (1967); ibid., 28, 343 (1968); and W. D. Odell and W. H. Daughaday, Principles of Competitive Binding Assays, Philadelphia: Lippincott Co., 1971.
Radio-immunoassay has a number of disadvantages. First of all, it requires the use of radioactive materials which pose potential health hazards and require special precautions for handling, as well as the observance of various licensing requirements. Secondly, the shelf life of radioactive reagents is very limited (usually about 30 days), due in part to the decay of radioactivity, and in part to the radio-decomposition of materials which are packaged with the radioactive label or tracer. Thirdly, standard curves for a given set of reagents change rapidly with time and must be redetermined frequently. Fourthly, the separation of bound from free antigen or ligand often is tedious and difficult. Fifthly, the measurement of radioactivity is complex, time-consuming, and does not lend itself to automation. Lastly, in spite of its great sensitivity, simple radio-immunoassays may not be sufficiently sensitive for the assay of many antigens or ligands present in truly small amounts.
In order to overcome some of these disadvantages, assays have been developed in which the known antigen or ligand is labeled with an enzyme instead of with a radioactive atom. After separation of bound and free ligand, the amount of known ligand or antigen in the free phase is quantified by measurement of enzyme activity, usually by a colorimetric reaction. Such so-called enzyme-immunoassays are described by L. E. M. Miles and C. N. Hales, Nature 219, 186 (1968); E. Engvall, and P. Perlman, Immunochemistry 8, 871 (1971); A. H. W. M. Schuurs and B. K. van Weemen, U.S. Pat. No. 3,654,090; and A. H. W. M. Schurrs and B. K. van Weemen, U.S. Pat. No. 3,838,153. Because one molecule of enzyme catalyzes the conversion of many molecules of substrate to product, such enzyme-immunoassays frequently provide "amplification", and therefore higher sensitivity than comparable radio-immunoassays.
To date, in all examples of enzyme immunoassays described in the scientific or patent literature, the activity of enzyme attached to antigen or ligand is inhibited when combined with antibody or binding protein. The inhibition of enzyme activity in the bound phase prevents quantification of antigen or ligand in the bound phase, a serious shortcoming that has made it difficult to develop workable assay systems using fluorescent markers. However, the inhibition of enzyme activity in the bound phase has the advantage that it lowers the enzyme activity in the whole assay mixture. This has been utilized to develop homogeneous enzyme-immunoassays in which there is no need to separate bound from free phases because of the inhibition of the bound phase. Such homogeneous assays are described by K. E. Rubenstein and E. F. Ullman, U.S. Pat Nos. 3,817,837, 3,852,157, and 3,975,237.
While the development of enzyme-immunoassays and homogeneous enzyme-immunoassays have overcome some of the shortcomings of radio-immunoassays listed above, some disadvantages remain. It would be highly desirable, therefore, to have fluorescent assays, particularly fluorescent immunoassays, which could provide sensitivity the same or greater than radioactive assays and radio-immunoassays, so that the disadvantages of using radioactive materials could be avoided. Such fluorescent immunoassays would be even more desirable if they were simple to perform, accurate, and lent themselves to automation. Similarly, it would be advantageous to have other highly sensitive assays using light emitting substances in place of radio-active tracers. The use of fluorogenic substrates, i.e., nonfluorescent compounds that enzymes convert to fluorescent products, is well known to those schooled in the art as a means of assaying enzyme activity and many such fluorogenic substrates are commercially marketed. However, their application to the assay of enzyme activity in biological samples or in immunoassays has been generally limited by unpredictable fluorescence or quenching in the sample which interferes with fluorescence quantification.